Emerging evidence suggests that connexins and gap junctional intercellular communication (GJIC) play a critical role in bone turnover especially in response to mechanical load. In vitro studies suggest that GJIC is critical for maximal bone cell network response to mechanical load and predict that, in the absence of GJIC, bone would be less responsive to mechanical load. However, recent in vivo data from our laboratory, as well as others, suggest that, quite paradoxically, deficiency in bone cell connexin 43 (Cx43), the predominant gap junction protein in bone, actually increases bone's anabolic response to mechanical load. Furthermore, our laboratory as well as one other has recently demonstrated that Cx43 deficiency desensitize bone to the catabolic effect of mechanical unloading. These recent developments suggest a paradigm shift in our understanding of connexins, GJIC and mechanotransduction in bone. That is, inhibiting bone cell Cx43 expression or GJIC has a beneficial effect on bone's response to its mechanical environment. However, the mechanism underlying this surprising development is unknown. To address this we will examine the hypothesis that inhibiting osteocytic Gja1 expression increases the anabolic response of bone to mechanical load and decreases the catabolic response to unloading through a mechanism involving apoptosis, RANKL/OPG and WNT/-catenin signaling pathways. During this 5 year project we will use in vitro cell culture and well established, as well as newly developed, genetically engineered in vivo mouse models, to examine 3 specific aims: 1) Examine the effect of mechanical loading on bone from osteocyte selective Cx43-deficient mice and mice with osteocytes deficient in both Cx43 and -catenin; 2) Examine the effect of mechanical unloading via hind limb suspension (HLS) on bone from the same genotypes examined in aim 1; and 3) Examine the effect of fluid flow on WNT/-catenin and RANKL/OPG signaling in osteocytic cells with and without Cx43. The expected outcomes of our research will contribute molecular-level knowledge of bone adaptation to its mechanical environment, provide seminal mechanistic insights into metabolic bone disease and facilitate development of new skeletal regeneration strategies, thereby providing a broad translational basis for our research focus.